Nicotinic acetylcholine receptors are ligand-gated ion channels which play a part in neurotransmission in the animal kingdom. The binding of acetylcholine or other agonists to the receptor causes a transient opening of the channel and allows cations to flow through. It is assumed that a receptor consists of five subunits grouped around a pore. Each of these subunits is a protein consisting of an extracellular N-terminal part followed by three transmembrane regions, an intracellular part, and a fourth transmembrane region and a short extracellular C-terminal part. Certain subunits carry on their extracellular part the binding site for ligands such as acetylcholine. Two vicinal cysteines form part of this binding site and are therefore a structural feature common to all ligand-binding subunits, which are also referred to as α subunits. Subunits without this structural feature are referred to, depending on the localization and function of the receptor, as β, γ, δ or ε subunits (Changeux et al. 1992).
Acetylcholine receptors have been particularly well investigated in vertebrates. Three groups can be distinguished on the basis of their anatomical localization and their functional properties (conduction properties of the channel, desensitization, sensitivity to agonists and antagonists and to toxins such as, for example, α-bungarotoxin). The classification correlates with the molecular composition of the receptors. They are heterooligomeric receptors with the subunit composition α2βγδ, which occur in muscle (Noda et al. 1982, Claudio et al. 1983, Devillers-Thiery et al. 1983, Noda et al. 1983a, b), heterooligomeric receptors which contain subunits from the α2–α6 and β2–β4 group and which occur in the nervous system (Schoepfer et al. 1990, Heinemann et al. 1997) and homooligomeric receptors which contain subunits from the α7–α9 group and which likewise occur in the nervous system (Lindstrom et al. 1997, Elgoyhen et al. 1997). This classification is also supported by the relationship of the gene sequences of the various subunits. The sequences of functionally homologous subunits from different species are typically more similar than sequences of subunits from different groups but from the same species. This is illustrated with some examples in FIG. 1B. In addition, the gene sequences of all known acetylcholine receptor subunits are similar to a certain extent not only with one another but also with those of some other ligand-gated ion channels (for example the serotonin receptors of the 5HT3 type, the GABA-gated chloride channels, the glycine-gated chloride channels). It is therefore assumed that all these receptors are derived from a common precursor and they are assigned to a gene superfamily (Ortells et al. 1995).
In insects, acetylcholine is the most important excitatory neurotransmitter in the central nervous system. Accordingly, acetylcholine receptors can be detected electrophysiologically in preparations of central ganglia from insects. Detection is possible both at postsynaptic and presynaptic nerve endings and on the cell bodies of interneurons, motor neurons and modulatory neurons (Breer et al. 1987, Buckingham et al. 1997). Among the receptors there are some which are inhibited by α-bungarotoxin and some which are insensitive (Schloβ et al. 1988). The acetylcholine receptors are moreover the molecular point of attack of important natural (for example nicotine) and synthetic insecticides (for example chloronicotinyls).
The gene sequences of a number of insect nicotinic acetylcholine receptors are already known. Thus, the sequences of five different subunits in Drosophila melanogaster have been described (Bossy et al. 1988, Hermanns-Borgmeyer et al. 1986, Sawruk et al. 1990a, 1990b, Schulz et al. 1998), likewise five in Locusta migratoria (Hermsen et al. 1998), one in Schistocerca gregaria (Marshall et al. 1990), six in Myzus persicae (Sgard et al. 1998, Huang et al. 1999), two sequences in Manduca sexta (Eastham et al. 1997, Genbank AJ007397) and six in Heliothis virescens (Genbank AF 096878, AF 096879, AF 096880, AF143846, AF143847, AJ 000399). In addition, a number of partial gene sequences from Drosophila melanogaster has been characterized as so-called expressed sequence tags (Genbank AA540687, AA698155, AA697710, AA697326). All these sequences are classified into α and β subunits depending on whether the two vicinal cysteines are present in the ligand binding site or not.
Recombinant expression of insect nicotinic receptors has proved to be more difficult than that of the analogous receptors from vertebrates or C. elegans. Thus, it has not yet been possible to express nicotinic receptors consisting only of insect subunits in such a way that their functional properties are the same as those of natural receptors (Marshall et al. 1990, Amar et al. 1995, Hermsen et al. 1998, Sgard et al. 1998). Relevant functional properties are, for example, sensitivity to agonists and antagonists, conductance for ion currents or desensitization. However, at least some α subunits from various insect species contribute to a functional receptor on coexpression of a vertebrate non-α subunit in place of an insect β subunit. The ligand-induced conductance of such hybrid receptors has been investigated in Xenopus laevis oocytes. Combinations of, for example, the Drosophila α1, α2 or α3 subunit with the chicken or rat β2 subunit lead to receptors whose sensitivity to agonists and antagonists or whose conductance for ion currents resemble those receptors detected in native preparations (Bertrand et al. 1994, Lansdell et al. 1997, Schulz et al. 1998, 2000, Matsuda et al. 1998). On the other hand, it has to date been possible to detect the expression in cell lines of hybrid receptors consisting, for example, of combinations of the Myzus persicae α1, α2 or α3 subunit with the rat β2 subunit or of combinations of the Drosophila α1, α2 or α3 subunit with the rat β2 or β4 subunit only through the binding of nicotinic ligands (Lansdell et al. 1997, 2000, Huang et al. 1999). The ligand-induced conductance of such receptors has not to date been detected in any case.
A further attempt to approach the expression of insect nicotinic receptors is represented by chimeric subunits (van den Beukel 1998). Sections of the gene sequence of the Drosophila α2 subunit were inserted recombinantly into the gene sequence of the rat α7 subunit. Expression of the chimeras in Xenopus laevis oocytes was detectable through binding of nicotinic ligands, but these receptors did not display the ligand-induced conductance either.